Morphological evidence that the molecularly determined Ciona intestinalis type A and type B are different species: Ciona robusta and Ciona intestinalis

Ciona intestinalis is considered a widespread and easily recognizable tunicate, the sister group of vertebrates. In recent years, molecular studies suggested that C. intestinalis includes at least two cryptic species, named ‘type A’ and ‘type B’, morphologically indistinguishable. It is dramatic to certify that two different species may be hidden under the name of a species widely used as a model species in biological researches. This raised the problem of identifying diagnostic morphological characters capable of distinguishing these types. We compared the morphology of specimens belonging to the two types and found that only type A specimens possess tunic tubercular prominences, allowing unambiguous discrimination. Remarkably, these structures were already described as distinctive of the Japanese species Ciona robusta, Hoshino and Tokioka, 1967; later synonymized under C. intestinalis (sensu Millar, 1953). In this study, we have confirmed that C. intestinalis type A corresponds to C. robusta. Based on the geographic distribution of C. intestinalis type B, and considering that the original C. intestinalis species was described from North European waters, we determined that C. intestinalis type B corresponds to C. intestinalis as described by Millar in 1953 and possibly to Linnaeus' Ascidia intestinalis L., 1767 for which we have deposited a neotype (from Roscoff, France) and for which we retain the name Ciona intestinalis (Linnaeus, 1767).     

Mechanism of the block to hybridization and selfing between the sympatric ascidians Ciona intestinalis and Ciona savignyi

The solitary ascidians Ciona intestinalis and Ciona savignyi co-occur in southern California harbors, but no hybrids have been recognized in nature. Numerous differences in their egg morphology were detected. Homologous (normal outcross) fertilization yielded 96-99% cleavage, where autologous (self) fertilization showed 3% and heterologous (hybrid) fertilization showed 0-1%. Acid treatment (pH 3.2) removed the block to selfing (P < 0.0001) but not hybridization for both species. Heterologous sperm bind to the vitelline coat (VC), but fail to penetrate. Enzymatic removal of the VC resulted in 91-97% cleavage with autologous and heterologous sperm (P < 0.0001). The vitelline coats of the two species differ in lectin binding to surface glycosides. Fertilization in both species is significantly inhibited by the lectins, fucose binding protein (P < 0.0001) and concanavalin A (P < 0.0001), and wheat germ agglutinin inhibits fertilization in C. intestinalis (P < 0.0001) but is without effect on C. savignyi fertilization. Self and hybrid blocks employ different mechanisms including glycoside composition and acid sensitivity.     

Immunological Maturation in the Tunicate Ciona intestinalis

The least explored areas in the phylogenetic development ofimmunity are the primitive chordate subphyla. The limited studieson tunicate internal defense mechanisms are presented and discussed.Primary and secondary immune responses and immunological maturationto vertebrate erythrocytes in Ciona intestinalis suggest thatits internal defense mechanisms may be closely allied to boththe invertebrates and vertebrates.     

Genetic and Genomic Toolbox of the Chordate Ciona intestinalis

The experimental malleability and unique phylogenetic position of the sea squirt Ciona intestinalis as part of the sister group to the vertebrates have helped establish these marine chordates as model organisms for the study of developmental genetics and evolution. Here we summarize the tools, techniques, and resources available to the Ciona geneticist, citing examples of studies that employed such strategies in the elucidation of gene function in Ciona. Genetic screens, germline transgenesis, electroporation of plasmid DNA, and microinjection of morpholinos are all routinely employed, and in the near future we expect these to be complemented by targeted mutagenesis, homologous recombination, and RNAi. The genomic resources available will continue to support the design and interpretation of genetic experiments and allow for increasingly sophisticated approaches on a high-throughput, whole-genome scale.     

Early oogenesis in the ascidian Ciona savignyi

Summary

Morphology of the germinal epithelium and the early follicular oocyte in the ascidian Ciona savignyi as examined by electron microscopy. The oogenetic part of the germinal epithelium contains oocytes at two different stages and the dark and clear cells. The smaller oocyte contains synaptonemal complexes. The larger oocyte in the initial phase of growth has a conspicuous nucleolus, electron-dense materials and some mitochondria close to the nuclear envelope. The nucleus of the larger oocyte is round and has the smooth contour. The dark cell contains a relatively large nucleus and is sometimes connected to each other by an intercellular bridge. Therefore, the dark cell, which has been suggested to be the progenitor cell of two kinds of accessory cells, may be also the oogonium. The early follicular oocyte just after migration from the germinal epithelium retains most of cytological features similar to those of the larger oocyte. However, the nuclear contour of the early follicular oocyte is uneven. This difference in the nuclear contour probably indicates that such a follicular oocyte is in the second phase of growth.     

Studies on the receptors in Ciona intestinalis

Summary A photoreceptor type structure not previously described has been found in the dorsal wall of the cerebral vesicle of the tadpole larva of Ciona intestinalis. The membranes of this receptor are organised as tubules some 60–100 nm in diameter and up to 1.5 m long. The tubules are confined in bundles about 1.5 m in diameter, which extend from the cell surface into the cavity of the cerebral vesicle. These tubules are similar to those in the rhabdomeric type of photoreceptor. However, in the cells from which the tubule processes arise are structures typical of the bases of cilia, and found in ciliary type photoreceptors.I should like to thank Professor J. Z. Young, F. R. S. for his continuing encouragement and help, and Dr. R. Bellairs for the use of electron microscope facilities. Mr. R. Moss and Mrs. J. Hamilton gave excellent technical assistance.     

Self- and cross-fertilization in the solitary ascidian Ciona savignyi

Solitary ascidians are hermaphrodites that release sperm and eggs simultaneously. However, many species are self-sterile, owing to a self/non-self recognition system operating at the outer surface of the chorion during sperm-egg interaction. In Ciona intestinalis, self-incompatibility is thought to have a genetic basis. Here, we report a survey of the self-fertilization potential of a Santa Barbara, California, population of Ciona savignyi, a close relative of C. intestinalis. We found that, in contrast to reports on C. intestinalis, C. savignyi is highly self-fertile. However, using two nonlethal recessive mutant strains, aimless (aim) and immaculate (imc), and a stable transgenic strain that expresses green fluorescent protein (GFP) in the notochord to follow offspring genotype, we demonstrate that non-self sperm outcompete self-sperm in fertilization competition assays. When the chorion was removed, both self- and non-self sperm performed equally well in the competition assay. Thus the non-self/self gamete recognition in C. savignyi is not absolute but relative, and is mediated by one or more components in the chorion. We discuss the significance of this finding in the context of natural populations in the wild, where individuals of C. savignyi are typically found growing in large groups that spawn in unison and where self-fertilization would be expected to be very rare.     

Retroduplication and loss of parental genes is a mechanism for the generation of intronless genes in Ciona intestinalis and Ciona savignyi

Tunicates, the sister clade of vertebrates, have miniature genomes and numerous intronless genes compared to other animals. It is still unclear how the tunicates acquired such a large number of intronless genes. Here, we analyzed sequences and intron–exon organizations of homologous genes from two closely related tunicates, Ciona intestinalis and Ciona savignyi. We found seven cases in which ancestral introns of a gene were completely lost in a species after their divergence. In four cases, both the intronless copy and the intron-containing copy were present in the genome, indicating that the intronless copy was generated by retroduplication. In the other three cases, the intron-containing copy was absent, implying it was lost after retroduplication. This result suggests that retroduplication and loss of parental genes is a major mechanism for the accumulation of intronless genes in tunicates.     

Identification of Early Oogenetic Cells in the Solitary Ascidians, Ciona savignyi and Ciona intestinalis: An Immunoelectron Microscopic Study

We raised monoclonal antibodies against homogenates of ovaries of Ciona intestinalis . We obtained an antibody named GC-1 which specifically recognized early germ cells in C. intestinalis and C. savignyi . Using GC-1 as a marker in immunoelectron microscopy, we determined the morphological sequence of early oogenesis in the ovaries of Ciona . In the stratified epithelium composing the wall of the ovarian tubes, the oocytes were identifiable at the early stages of meiotic prophase according to nuclear features such as condensed chromatin with synaptonemal complexes. GC-1 recognized these early oocytes. We found round cells with large and homogeneous nuclei clustered at the marginal end of the stratified epithelium. We identified these cells as oogonia on the basis of: (1) features of the nucleus, (2) reactivity to GC-1, and (3) early emergence in the developing ovaries. The oogonia were classified into three types: type A was large (7–9 μm in diameter) and clear, type B was intermediate in size (5–6 μm) and electron-density, and type C was small (4–5 μm) and dark. In the developing ovaries of juvenile C. intestinalis, type A oogonia appeared first (before 11 days after settlement) and types B and C followed (15 days after settlement). Thus we see that the type A is the oogenetic stem cell, type B is the proliferating oogonium, and type C is the final oogonium just before meiosis. The oocytes appeared 18 days after metamorphosis.     

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing.     

Acrosome differentiation in the ascidians Clavelina lepadiformis and Ciona intestinalis

The spermatozoa of both Clavelina lepadiformis and Ciona intestinalis have architectural features characteristic of ascidian spermatozoa that have been previously described. They have an elongated head (6 microm and 3 microm long, respectively) and a single mitochondrion that is closely applied laterally to the nucleus; they lack a midpiece. The acrosome of Clavelina lepadiformis spermatozoa is a moderately electron-dense, pear-shaped flattened vesicle, approx. 300 nm x 200 nm x 40 nm in length, width, and height, respectively. The acrosome of Ciona intestinalis spermatozoa is a moderately electron-dense, round flattened vesicle with an electron-dense plate in its central region. It is approx. 200 nm x 200 nm x 50 nm in length, width, and height, respectively. During spermiogenesis in both ascidians, several proacrosomal vesicles (50-70 nm in diameter) appear in a blister at the future apex of the spermatids. These vesicles appear to be associated with the inner surface of the plasma membrane enclosing the blister. They come into contact with each other along the inner surface of the plasma membrane and fuse to form a horseshoe-shaped acrosomal vesicle, which becomes a round, flattened vesicle during further differentiation. Some speculations about the mechanism of acrosome differentiation, the possible role of the acrosome during fertilization, and in the speciation of ascidians are presented.     

An analysis of the genome of Ciona intestinalis

An analysis by CsCl density gradient centrifugation has shown that, at a fragment size of about 100 kb, the DNA of a urochordate, Ciona intestinalis, is remarkably homogeneous in base composition. Localization of 16 coding sequences from C. intestinalis, chosen so as to cover the distribution range of all available coding sequences for this organism, showed a nearly symmetrical distribution almost coinciding with the DNA distribution. Both distributions are remarkably different from those found in vertebrates, which are skewed towards high GC levels (to a greater extent in warm-blooded vertebrates). In order to account for this change in genome organization, we propose a working hypothesis that can be tested. Basically, we suggest that the genome duplication that occurred between urochordates and fishes was accompanied by a preferential integration of transposons in one compartment of the genome, which was made gene-poor (by lowering gene density) compared to the rest. Since the gene-poor compartment (the 'empty quarter') is characterized by a lower level of gene expression compared to the gene-rich compartment (the 'genome core') in the vertebrate genome, we further suggest, as a working hypothesis, that a compartmentalization according to gene expression already existed in urochordates.     

Action of morpholinos in Ciona embryos

First, using morpholino against lacZ, we demonstrate that the morpholino specifically suppresses the translation of the gene introduced exogenously into Ciona eggs. Second, using morpholino against an alkaline phosphatase gene, we show that the morpholino suppresses the translation of the endogenous gene as well. Third, using morpholino against beta-catenin gene, we confirm that the suppression by the morpholino can be rescued by injection of beta-catenin mRNA. All of these results indicate that morpholino act in Ciona embryos to specifically block the function of endogenous genes as well as exogenously introduced genes. genesis 30: 103--106, 2001.     

Computational analysis of Ciona intestinalis operons

Operons are clusters of genes that are co-regulated from a common promoter. Operons are typically associated with prokaryotes, although a small number of eukaryotes have been shown to possess them. Among metazoans, operons have been extensively characterized in the nematode Caenorhabditis elegans in which ～15% of the total genes are organized into operons. The most recent genome assembly for the ascidian Ciona intestinalis placed ～20% of the genes (2909 total) into 1310 operons. The majority of these operons are composed of two genes, while the largest are composed of six. Here is reported a computational analysis of the genes that comprise the Ciona operons. Gene ontology (GO) terms were identified for about two-thirds of the operon-encoded genes. Using the extensive collection of public EST libraries, estimates of temporal patterns of gene expression were generated for the operon-encoded genes. Lastly, conservation of operons was analyzed by determining how many operon-encoded genes were present in the ascidian Ciona savignyi and whether these genes were organized in orthologous operons. Over 68% of the operon-encoded genes could be assigned one or more GO terms and 697 of the 1310 operons contained genes in which all genes had at least one GO term. Of these 697 operons, GO terms were shared by all of the genes within 146 individual operons, suggesting that most operons encode genes with unrelated functions. An analysis of operon gene expression from nine different EST libraries indicated that for 587 operons, all of the genes that comprise an individual operon were expressed together in at least one EST library, suggesting that these genes may be co-regulated. About 50% (74/146) of the operons with shared GO terms also showed evidence of gene co-regulation. Comparisons with the C. savignyi genome identified orthologs for 1907 of 2909 operon genes. About 38% (504/1310) of the operons are conserved between the two Ciona species. These results suggest that like C. elegans, operons in Ciona are comprised of a variety of genes that are not necessarily related in function. The genes in only 50% of the operons appear to be co-regulated, suggesting that more complex gene regulatory mechanisms are likely operating.